Genetically engineered microbes and uses thereof

ABSTRACT

This invention concerns methods of identifying genetic alterations with which a microbe can be used to produce fatty acids at a large amount for making biofuels. Also disclosed are microbes with such genetic alterations and uses thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.61/447,746 filed on Mar. 1, 2011. The content of the application isincorporated herein by reference in its entirety.

FIELD OF INVENTION

This invention relates to genetically engineered microbes that can beused to produce fatty acids at a high level for making fuels and relatedmethods.

BACKGROUND OF INVENTION

The industrialized world has been relying on fossil fuels for centuriesto provide, among others, electricity, gasoline, jet fuel, plastics, andso on. As supplies of fossil fuels are limited, there is a critical needto develop alternative energy sources, including renewable energy.Microbes offer great promise to contribute a significant portion of therenewable energy.

Genome-scale flux-balance analysis (FBA) modeling has been shown to beuseful for the in silico design of engineered strains of microbes thatoverproduce diverse targets. These engineered strains includeEscherichia coli that overproduce lycopene (Alper et al. 2005 MetabolicEngineering 7(3): 155-164; and Alper et al. 2005 Nature Biotechnology23(5): 612-616), lactic acid (Fong et al. 2005 Biotechnology andBioengineering 91(5): 643-648), succinic acid (Lee et al. 2005 Appliedand Environmental Microbiology 71(12): 7880-7887; and Wang et al. 2006Applied Microbiology and Biotechnology 73(4): 887-894), L-valine (Parket al. 2007 Proceedings of the National Academy of Sciences, USA104(19): 797-7802), and L-threonine (Lee et al. 2007 Molecular SystemsBiology 3: 149) and strains of Saccharomyces cerevisiae that overproduceethanol (Bro et al. 2006 Metabolic Engineering 8(2): 102-111; andHjersted et al. 2007 Biotechnology and Bioengineering 97(5): 1190-1204).FBA models allow the result of various genetic manipulations strategiesto be predicted. As a result, the space of possible geneticmanipulations can be computationally searched for the strategy thatresults in the desired metabolic network state. This space is vast, andalgorithms must be designed to search the space efficiently.

Transforming bi-level optimization of FBA models to single levelmixed-integer linear programming (MILP) problems (Burgard et al. 2003Biotechnology and Bioengineering 84(6): 647-657; Pharkya et al. 2004Genome Research 14(11): 2367-2376; and Pharkya et al. 2006 MetabolicEngineering 8(1): 1-13) has resulted in computational methods thatefficiently search the space of genetic manipulations. This approach ismuch more efficient than exhaustive, brute-force search, but it isnevertheless very computationally intensive. The runtimes scaleexponentially as the number of manipulations allowed in the final designincreases. For large models, such as the latest genome-scale model of E.coli K-12 MG1655 (Feist et al. 2007 Molecular Systems Biology 3: 121),iAF1260, it was found that this runtime generally proves prohibitive fordesigns involving more than a few manipulations. Given the fact thatuseful metabolically engineered strains often require many geneticmanipulations (such as the artemisinic-acid-producing strain of S.cerevisiae by Ro et al. 2006, Nature 440(7086): 940-943), which requiredthe addition of three genes and the up- or down-regulation of fourgenes) and that the number of reactions, metabolites, and genes inmetabolic, models continues to grow (Feist et al. 2008 NatureBiotechnology 26(6): 659-667). There is a need for more efficientcomputational search techniques for effective in silico design.

SUMMARY OF INVENTION

This invention is based, at least in part, on an unexpected discovery ofa new methodology for identifying a target gene for a geneticalteration, where an engineered microbe having the genetic alterationproduces a fatty acid at a level higher than a control microbe thatlacks the genetic alteration.

Accordingly, one aspect of the invention features a method forconstructing a model for identifying a target gene for a geneticalteration. The method includes step of obtaining a genome-scaleflux-balance analysis (FBA) model; adding to the model a component thatmodels a thioesterase shunt, wherein the added component forces afraction of the flux passing through a fatty acyl-ACP compound in themodel through the following reaction:

fatty acyl-ACP[c]→ACP[c]+free fatty acid[e],

wherein [c] represents the cytoplasm compartment and [e] representsextracellular space; obtaining a biological optimal flux distribution;and displaying a record comprising a gene returned by the model, wherebythe gene is the target for the genetic alteration. An engineered microbehaving the genetic alteration produces a fatty acid at a level higherthan a control microbe that is identical to the engineered microbeexcept that the control microbe lacks the genetic alteration.

In a second aspect, the invention features a method for identifying atarget for a genetic alteration. An engineered microbe having thegenetic alteration produces a fatty acid at a level higher than acontrol microbe that is identical to the engineered microbe except thatthe control microbe lacks the genetic alteration. The method includesobtaining one or more genes using the model constructed by the methoddescribed above.

In the above methods, the microbe can be a bacterium (e.g., E. coli) ora yeast (e.g., Saccharomyces cerevisiae). The model can be an iAF1260model as describe in Alper et al. 2005 Metabolic Engineering 7(3):155-164; and Alper et al. 2005 Nature Biotechnology 23(5): 612-616; Fonget al. 2005 Biotechnology and Bioengineering 91(5): 643-648; Lee et al.2005 Applied and Environmental Microbiology 71(12): 7880-7887; Wang etal. 2006 Applied Microbiology and Biotechnology 73(4): 887-894; Park etal. 2007 Proceedings of the National Academy of Sciences, USA 104(19):797-7802; Lee et al. 2007 Molecular Systems Biology 3: 149; Bro et al.2006 Metabolic Engineering 8(2): 102-111; and Hjersted et al. 2007Biotechnology and Bioengineering 97(5): 1190-1204. These publicationsare incorporated in this application by reference in their entirety. Inone embodiment, the biological optimal flux distribution is obtained bya Genetic Design through Local Search (GDLS) approach. The GDLS can becarried out in the manner described in Lun et al. 2009 Molecular SystemsBiology 5: 296, the content of which is hereby incorporated by referencein its entirety. The genetic alteration can be an alteration of two ormore genes. The genetic alteration can be a knockout of a gene orover-expression of a gene.

In a third aspect, the invention features a machine-readable medium forcarrying out the methods described above. The machine-readable mediumhas machine-readable instructions encoded thereon which, when executedby a processor, cause a machine having or linked to the processor toexecute each of the methods.

In a fourth aspect, the invention features a machine-readable medium onwhich is stored a database capable of configuring a computer to respondto queries based on a plurality of records or values belonging to thedatabase. Each of the records has one or more of the following values:

a genotype value that identifies a genotype of a microbe having one ormore genetic alterations;

a gene value that identifies a gene having a genetic alteration;

a fatty acid value that identifies a fatty acid;

a biomass flux value that identifies a biomass flux; and

a free fatty acid flux value that identifies a free fatty acid flux.

The biomass flux and free fatty acid flux are obtained using the modelof methods described above.

This invention also features a computer system having theabove-mentioned machine-readable medium and a user interface capable ofreceiving the above-mentioned data and displaying the above-mentionedrecord.

In a fifth aspect, the invention features an isolated or cultured cellthat lacks a functional gene, wherein the gene is selected from thegroup consisting of fadE (e.g., GenBank Accession NO.: NP_(—)414756),rpe (e.g., GenBank Accession NO.: NP_(—)417845), sgcE (e.g., GenBankAccession NO.: NP_(—)418721), talA e.g., GenBank Accession NO.:(NP_(—)416959), and talB (e.g., GenBank Accession NO.: NP_(—)414549)genes and a homologue thereof. In one example, the cell lacks afunctional fadE gene (i.e., fadE gene knockout); in another, the celllacks both functional rpe/sgcE gene and talAB gene (i.e., adouble-knockout of these two genes). The cell can be a bacterium cell(e.g., E. coli) or a yeast cell (e.g., Saccharomyces cerevisiae).Preferably, the cell is an E. coli. cell, such as a K-12 MG1665 cell.The cell can be one expressing a thioesterase, such as E. colithioesterase gene tesA, 'tesA, or plant thioesterases as described inCho et al. 1995 J. Biol. Chem. 270(9): 4216-4219, Voelker et al. 1994 J.Bacteriol. 176(23): 7320-7327; Dehesh et al. 1996 The Plant Journal9(2): 167-172; and Steen et al. 2010 Nature 463(7280): 559-562). Thesepublications are incorporated by reference in their entirety.

In a sixth aspect, the invention features a method of producing a fattyacid. The method includes steps of culturing the above-described cell ina culture under conditions allowing producing of the fatty acid by thecell, and obtaining the fatty acid from the culture.

In a seventh aspect, the invention features a method of a hydrocarbonproduct. Briefly, fatty acids can be converted to jet fuel and gasolineby thermally decarboxylating fatty acids to form linear hydrocarbons,then hydrocracking and isomerizing the fatty acids to form branchedhydrocarbons in the jet fuel or gasoline range. The method can beconducted in the manner described in WO 2007027955 or US Application No.20080229654. These publications are incorporated by reference in theirentirety. For example, the method can include steps performing thermaldecarboxylation on fatty acids to form a thermal decarboxylation productstream; hydrocracking the thermal decarboxylation product stream, andisolating a product in the gasoline, jet, or diesel fuel range. At leasta portion of the fatty acids are derived from the cell described above.Also all or a portion of the product can be subjected to isomerizationconditions, hydrogenation, hydrotreatment, and/or hydrofinishingconditions.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims.

DESCRIPTION OF DRAWING

FIGS. 1A and B show results of genetic design through local searches forgenetic alteration strategies for fatty acid overproduction in E. coliK-12 MG1655 with thioesterase shunt.

FIG. 2 is a diagram showing an exemplary computer system.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides to methods and systems for identifyingcandidates of genetic alterations that result in fatty acidoverproduction in microbes, such as bacteria (e.g., E. coli). Fattyacids produced by these microbes can be used for producing biofuels. Theeconomical and sustainable production of liquid transportation fuelsthat are not derived from fossil fuels, particularly crude oil, is apressing problem concerning U.S. national interests and the world morebroadly. Lipid biofuels, including biodiesel, biogasoline, and biojetfuel, can be produced chemically or biologically from fatty acids. Thisinvention provides a novel computer modeling method that can be appliedto any metabolic engineering problem requiring the insertion of genesunder artificial transcriptional control. This invention also provides aspecific application of this method to the problem of producing fattyacids using Escherichia coli. As an example, mutant strains of E. coliK-12 MG1655, which were developed as a result of this modeling method,have significantly increased fatty acid production over the wild typestrain.

In one embodiment, this invention uses a two-stage process for producingjet fuel from food and non-food feed stocks. First, agenetically-engineered microorganism is used to convert the feedstock(e.g., various biomasses) into fatty acids. This organism is producedusing a computational design process to identify favorable geneticmodifications. Second, fatty acids are converted into jet fuel using achemical process. The jet fuel can be domestically produced and can beused by the aviation and defense industries.

Methodology

In one aspect, this application provides a highly efficient analysismethodology for identifying candidates of genetic alterations thatresult in fatty acid overproduction in microbes. The methodology isbased, at least in part, on an efficient computational method for insilico design—GDLS (Genetic Design through Local Search, Lun et al. 2009Molecular Systems Biology 5: 296). GDLS is a scalable heuristicalgorithmic method that employs an approach based on local search withmultiple search paths which results in effective, low-complexity searchof the space of genetic manipulations. Thus, GDLS is able to findgenetic designs with greater in silico production of desired metabolitesthan can be feasibly found using globally-optimal search and performsfavorably in comparison to heuristic searches based on evolutionaryalgorithms and simulated annealing.

As disclosed herein, GDLS can be applied to find genetic designstrategies for overproducing hydrocarbon such as acetate and succinateusing E. coli, which yielded results that were consistent with previousexperimental studies. These compounds—acetate and succinate—arenaturally produced and secreted by E. coli, and the design strategiesdescribed herein improve the efficiency of converting the feedstock,glucose, into the desired compound by linking the organism's biomassproduction with its production of the desired compound.

There are also many metabolites of interest that are not naturallyproduced and secreted by E. coli or other industrially attractivemicrobe. In these cases, exogenous genes must be introduced to cause thedesired compound to be produced and secreted. This is the case withfatty acid production using E. coli. E. coli cells naturally producefatty acids to make phospholipids that are incorporated into cellularcomponents, but the fatty acids are not naturally secreted into thegrowth medium. Because there is no natural metabolic sink for fattyacids, it is difficult to substantially increase their production. Byintroducing either a modified version of the E. coli thioesterase geneteas (Cho et al. 1995 J. Biol. Chem. 270(9): 4216-4219) 'tesA, orcertain plant thioesterases (Voelker et al. 1994 J. Bacteriol. 176(23):7320-7327; Dehesh et al. 1996 The Plant Journal 9(2): 167-172; and Steenet al. 2010 Nature 463(7280): 559-562) into E. coli, however, theorganism will secrete free fatty acids, and the production of fattyacids becomes decoupled from synthesis of cellular components, allowingfor overproduction.

The likely mechanism of these thioesterases is that they disrupt thenatural fatty acid biosynthesis pathway, where fatty acids are bound toACPs (acyl carrier proteins) and form fatty acyl-ACPs. The thioesterasescleave the fatty acids from the ACPs during this elongation process,leaving free fatty acids that then diffuse out of the cell into thesurrounding media Cho et al. 1995 J. Biol. Chem. 270(9): 4216-4219.

These thioesterases are genetically introduced using artificialpromoters that are induced or constitutively expressed. Therefore, theyare not under the natural regulatory control of the organism, and theyform an artificial metabolic “shunt”. This poses a problem for FBA,which assumes that, under certain constraints imposed on thecapabilities of the organism, most importantly the stoichiometricconstraints imposed by the enzymes it is capable of producing, theorganism regulates expression so as to achieve its biological objective,which is typically assumed to be the maximization of biomass production.

The present invention provides a method for modeling such artificialshunts by extending FBA. By coupling this method with computationalgenetic design methods such as GDLS, one can obtain genetic designstrategies for organisms containing such shunts. As shown in the examplebelow, this approach was applied to the problem of overproducing fattyacids using E. coli with a thioesterase under artificial regulatorycontrol, and it was shown that genetic designs obtained using themethodology of this invention resulted in overproduction of free fattyacids.

As shown in the example below, the methodology successfully identifiedgenetic alterations that can be used to increase fatty acid production.The methodology allows microbes, such as E. coli and other bacteria, tobe engineered for high-efficiency production of fatty acids. Whilebacteria can already be engineered to produce fatty acids, the greaterthe efficiency of the process in terms of its ability to produce highyields of fatty acids for a given amount of feedstock, the cheaper theprocess.

Computer Products, Systems, and Instruments

The computational methodology of this invention can be incorporated intoa multiplicity of suitable computer products, systems, and/orinformation instruments. User interface methods known in the informationprocessing art can be used in the systems of this invention.

1. Computer Software

For example, the above-disclosed methodology or components thereof canbe embodied in a fixed medium (e.g., a computer accessible/computerreadable medium program component containing logic instructions or data,or both), that when loaded into an appropriately configured computingdevice can cause that device to perform operations to the invention. Invarious embodiments a fixed medium component containing logicinstructions can be delivered to a viewer on a fixed medium forphysically loading into a viewer's computer or a fixed medium containinglogic instructions can reside on a remote server that a viewer canaccess through a communication medium in order to download a programcomponent.

Examples of a tangible computer-readable medium suitable for usecomputer program products and computational apparatus of this inventioninclude, but are not limited to, magnetic media such as hard disks,floppy disks, and magnetic tape; optical media such as CD-ROM disks;magneto-optical media; semiconductor memory devices (e.g., flashmemory), and hardware devices that are specially configured to store andperform program instructions, such as read-only memory devices (ROM) andrandom access memory (RAM) and sometimes application-specific integratedcircuits (ASICs), programmable logic devices (PLDs) and signaltransmission media for delivering computer-readable instructions, suchas local area networks, wide area networks, and the Internet. The dataand program instructions provided herein may also be embodied on acarrier wave or other transport medium (including electronic oroptically conductive pathways). The data and program instructions ofthis invention may also be embodied on a carrier wave or other transportmedium (e.g., optical lines, electrical lines, and/or airwaves).

2. Database

The above-described data and information generated using the methodologyof this invention can be used to establish a database, i.e., acollection of data, which can be used to analyze and respond to queries.In one embodiment, the database includes one or more records fororganizing the raw data sets and information sets in a particularhierarchy or directory (e.g., a hierarchy of studies and projects). Inaddition, the database may include information correlating the recordsto one another, a list of globally unique terms or identifiers for celllines, genes, chemical or metabolism reactions, or other features. Sucha database also contains a taxonomy that contains a list of all tags(keywords) for different genotypes, phenotypes, cells, as well as theirrelationships.

In one embodiment, the database contains data from a number of sources,including data from external sources, such as public databases). Inaddition, the database can include proprietary data obtained andprocessed by the database developer or user. A database may be updatedby a developer or user as new public or private information frombiological or chemical experiments becomes available. Once imported, alldata are correlated with other information in the database so as toenable users to interrogate cell lines, genes, chemical or metabolismreactions, genotypes, phenotypes, as well as their relationships acrossthe entire information space.

3. Computer Hardware

In another aspect, the invention provides an apparatus for performingthe above-mentioned operations. This apparatus may be specially designedand/or constructed for the required purposes, or it may be ageneral-purpose computer selectively configured by one or more computerprograms and/or data structures stored in or otherwise made available tothe computer. The processes presented herein are not inherently relatedto any particular computer or other apparatus. In particular, variousgeneral-purpose machines may be used with programs written in accordancewith the teachings herein, or it may be more convenient to construct amore specialized apparatus to perform the required method steps.

FIG. 2 illustrates an exemplary computer system (200) that, whenappropriately configured or designed, can serve as a computationalapparatus according to certain embodiments. The computer system 200includes any number of processors 202 (i.e., central processing units,or CPUs) that are coupled to storage devices including primary storage206 (typically a random access memory, or RAM), primary storage 204(typically a read only memory, or ROM). CPU 202 may be of various typesincluding microcontrollers and microprocessors such as programmabledevices (e.g., CPLDs and FPGAs) and non-programmable devices such asgate array ASICs or general-purpose microprocessors. In the depictedembodiment, primary storage 204 acts to transfer data and instructionsuni-directionally to the CPU and primary storage 206 is used typicallyto transfer data and instructions in a bi-directional manner. Both ofthese primary storage devices may include any suitable computer-readablemedia such as those described above. A mass storage device 208 is alsocoupled bi-directionally to primary storage 206 and provides additionaldata storage capacity and may include any of the computer-readable mediadescribed above. Mass storage device 208 may be used to store programs,data and the like and is typically a secondary storage medium such as ahard disk. Frequently, such programs, data and the like are temporarilycopied to primary memory 206 for execution on CPU 202. The informationretained within the mass storage device 208, may, in appropriate cases,be incorporated in standard fashion as part of primary storage 204. Aspecific mass storage device such as a CD-ROM 612 may also pass datauni-directionally to the CPU or primary storage.

CPU 202 is also coupled to an interface 610 that connects to one or moreinput/output devices such as video monitors, track balls, mice,keyboards, microphones, touch-sensitive displays, transducer cardreaders, magnetic or paper tape readers, tablets, styluses, voice orhandwriting recognition peripherals, USB ports, or other well-knowninput devices such as other computers. Finally, CPU 202 optionally maybe coupled to an external device such as a database or a computer ortelecommunications network using an external connection as showngenerally at network 614. With such a connection, it is contemplatedthat the CPU might receive information from the network, or might outputinformation to the network in the course of performing the method stepsdescribed herein.

In one embodiment, a system such as computer system 200 is used as aspecial purpose data import, data correlation, and querying systemcapable of performing some or all of the tasks described herein. System200 may also serve as various other tools associated with databasedescribed above and querying such as a data capture tool. Informationand programs, including data files can be provided via a networkconnection 214 for access or downloading from server system 216.Alternatively, such information, programs and files can be provided tothe researcher on a storage device. In a specific embodiment, thecomputer system 200 is directly coupled to a data acquisition systemsuch as a high-throughput screening system that captures data fromsamples. Data from such systems are provided via interface 210 foranalysis by system 200. Alternatively, the data processed by system 200are provided from a data storage source such as a database or otherrepository of relevant data. Once in apparatus 200, a memory device suchas primary storage 206 or mass storage 208 buffers or stores, at leasttemporarily, relevant data. The memory may also store various routinesand/or programs for importing, analyzing and presenting the data,including importing the above-described data sets, correlating data setswith one another and with feature groups, generating and runningqueries, etc.

In certain embodiments, the system of this invention may include one ormore user terminals (218). User terminals can include any type ofcomputer (e.g., desktop, laptop, tablet, etc.), media computingplatforms (e.g., cable, satellite set top boxes, digital videorecorders, etc.), handheld computing devices (e.g., PDAs, e-mailclients, etc.), cell phones or any other type of computing orcommunication platforms. A server (216) in communication with a userterminal may include a server device or decentralized server devices,and may include mainframe computers, mini computers, super computers,personal computers, or combinations thereof. A plurality of serversystems may also be used without departing from the scope of the presentinvention. User terminals and a server system may communicate with eachother through the network 214. The network may comprise, e.g., wirednetworks such as LANs (local area networks), WANs (wide area networks),MANs (metropolitan area networks), ISDNs (Intergrated Service DigitalNetworks), etc. as well as wireless networks such as wireless LANs,CDMA, Bluetooth, and satellite communication networks, etc. withoutlimiting the scope of the present invention.

Genetically Engineered Cells and Production of Fatty Acid

The above-described computational methodology successfully identifiedgenetic alterations that can be used to increase fatty acid production.Accordingly, microbes, such as E. coli and other bacteria, can beengineered to have those alterations and are useful for high-efficiencyproduction of fatty acids.

In one aspect, the invention provides a method of producing a fattyacid. The method includes culturing the above-mentioned geneticallyengineered host cell in the presence of a carbon source. In one example,the host cell is genetically engineered to lack one or more of a numberof functional genes or to have a reduced expression levels of the one ormore genes as compared to wildtype cells. Examples of these genesinclude fadE, rpe, sgcE, talA and talB. The host cell can also beengineered to over-express a gene encoding a thioesterase, such as tesA,'tesA, tesB, fatB, fatB2, fatB3, fatA1 . In some embodiments, the methodfurther comprises isolating the fatty acid.

In some embodiments, the fatty acid is present in the extracellularenvironment. In that case, the fatty acid can be isolated from theextracellular environment of the host cell using techniques known in theart. In some embodiments, the fatty acid is spontaneously secreted,partially or completely, from the host cell. In alternative embodiments,the fatty acid is transported into the extracellular environment,optionally with the aid of one or more suitable transport proteins. Inother embodiments, the fatty acid is passively transported into theextracellular environment.

In some embodiments, the host cell is cultured in a culture mediumcontaining an initial concentration of the carbon source of about 0.2%to 10% (w/v) or 2 g/L to about 100 g/L (e.g., about 2 to 10, 10 to 20,20 to 30, 30 to 40, 40 to 50 g/L) of a carbon source. In exemplaryembodiments, the culture medium contains an initial concentration ofabout 2 g/L or more of a carbon source. To that end, the method canfurther include a step of monitoring the level of the carbon source inthe culture medium. In some embodiments, the method further includesadding a supplemental carbon source to the culture medium when the levelof the carbon source in the medium is less than about 0.5 g/L (e.g.,0.4, 0.3, 0.2, or 0.1 g/L).

The methods disclosed herein can be performed using glucose as a carbonsource. In that case, microorganisms can be grown in a culture mediumcontaining an initial glucose concentration of about 2 g/L to about 50g/L, such as 20 g/L (i.e., 2% w/v). Since the glucose concentration ofthe medium decreases from the initial concentration as themicroorganisms consume the glucose, a concentration of about 0 g/L toabout 5 g/L glucose is maintained in the culture medium during the fattyester production process. Glucose can be fed to the microorganisms in asolution of about 50% to about 65% glucose. In some instances, the feedrate of glucose is set to match the cells' growth rate to avoid excessaccumulation of glucose. In certain embodiment, fatty acids can beproduced from carbohydrates other than glucose, including but notlimited to fructose, hydrolyzed sucrose, hydrolyzed molasses andglycerol.

Various cells can be used as the host cell in the above-describedmethod. Examples include a mammalian cell, plant cell, insect cell,yeast cell, fungus cell, filamentous fungi cell, and bacterial cell. Inone example, the host cell is selected from the genus Escherichia,Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus,Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces,Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus,Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas,Schizosaccharomyces, Yarrowia, or Streptomyces. In a preferredembodiment, the host cell is an E. coli cell. Various methods well knownin the art can be used to genetically engineer host cells to knockoutone or more genes or to over-express one or more of other genes.

As used herein, the term “deletion” or “knockout” means modifying orinactivating a polynucleotide sequence that encodes a target protein inorder to reduce or eliminate the function of the target protein. Apolynucleotide deletion can be performed by methods well known in theart (See, e.g., Datsenko et al., Proc. Nat. Acad. Sci. USA, 97:6640-45,2000 or International Patent Application Nos. PCT/US2007/011923 andPCT/US2008/058788). “Gene knockout,” as used herein, refers to aprocedure by which a gene encoding a target protein is modified orinactivated so to reduce or eliminate the function of the intactprotein. Inactivation of the gene may be performed by general methodssuch as mutagenesis by UV irradiation or treatment withN-methyl-N′-nitro-N-nitrosoguanidine, site-directed mutagenesis,homologous recombination, insertion-deletion mutagenesis, or “Red-drivenintegration” (Datsenko et al., Proc. Natl. Acad. Sci. USA, 97:6640-45,2000).

For example, in one embodiment, a construct is introduced into a hostcell, such that it is possible to select for homologous recombinationevents in the host cell. One of skill in the art can readily design aknock-out construct including both positive and negative selection genesfor efficiently selecting transfected cells that undergo a homologousrecombination event with the construct. The alteration in the host cellmay be obtained, for example, by replacing through a single or doublecrossover recombination a wild type DNA sequence by a DNA sequencecontaining the alteration. For convenient selection of transformants,the alteration may, for example, be a DNA sequence encoding anantibiotic resistance marker or a gene complementing a possibleauxotrophy of the host cell. Mutations include, but are not limited to,deletion-insertion mutations. An example of such an alteration includesa gene disruption, i.e., a perturbation of a gene such that the productthat is normally produced from this gene is not produced in a functionalform. This could be due to a complete deletion, a deletion and insertionof a selective marker, an insertion of a selective marker, a frameshiftmutation, an in-frame deletion, or a point mutation that leads topremature termination. In some instances, the entire mRNA for the geneis absent. In other situations, the amount of mRNA produced varies.

As used herein, “overexpress” means to express or cause to be expressedor produced a nucleic acid, polypeptide, or hydrocarbon in a cell at agreater concentration than is normally expressed in a correspondingwild-type cell. For example, a polypeptide can be “overexpressed” in arecombinant host cell when the polypeptide is present in a greaterconcentration in the recombinant host cell compared to its concentrationin a non-recombinant host cell of the same species.

For overexpression a polypeptide, the methods can include the use ofvectors, preferably expression vectors, containing a nucleic acidencoding a desired polypeptide, such as a thioesterase, as describedherein, polypeptide variant, or a fragment thereof. Those skilled in theart will appreciate a variety of viral vectors (for example, retroviralvectors, lentiviral vectors, adenoviral vectors, and adeno-associatedviral vectors) and non-viral vectors can be used in the methodsdescribed herein.

Expression of polypeptides in prokaryotes, for example, E. coli, canoften be carried out with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionpolypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino terminus of therecombinant polypeptide. Such fusion vectors typically serve threepurposes: (1) to increase expression of the recombinant polypeptide; (2)to increase the solubility of the recombinant polypeptide; and (3) toaid in the purification of the recombinant polypeptide by acting as aligand in affinity purification.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in, for example, Sambrook et al. Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989).

For stable transformation of bacterial cells, only a small fraction ofcells will take-up and replicate the expression vector depending uponthe expression vector and transformation technique used. In order toidentify and select these transformants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) can be introducedinto the host cells along with the gene of interest. Selectable markersinclude those that confer resistance to drugs, such as ampicillin,kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding aselectable marker can be introduced into a host cell on the same vectoras that encoding a polypeptide described herein or can be introduced ona separate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

With the above-described host cells and methods, one can produce variousfatty acids at concentrations shown in Table 2 below. The fatty acidproduced during fermentation can be separated from the fermentationmedia. Any known technique for separating fatty acids from aqueous mediacan be used. For example, one can used a two phase (bi-phasic)separation process. This process involves fermenting the geneticallyengineered host cells under conditions sufficient to produce a fattyacid, allowing the fatty acid to collect in an organic phase, andseparating the organic phase from the aqueous fermentation broth. Thismethod can be practiced in both a batch and continuous fermentationprocesses.

Once fatty acids are collected, they can be converted to jet fuel andgasoline by thermally decarboxylating fatty acids to form linearhydrocarbons, then hydrocracking and isomerizing the fatty acids to formbranched hydrocarbons in the jet fuel or gasoline range. The method canbe conducted in the manner described in WO 2007027955 or US ApplicationNo. 20080229654. These publications are incorporated by reference intheir entirety. For example, the method can include steps performingthermal decarboxylation on fatty acids to form a thermal decarboxylationproduct stream; hydrocracking the thermal decarboxylation productstream, and isolating a product in the gasoline, jet, or diesel fuelrange. At least a portion of the fatty acids are derived from the celldescribed above. Also all or a portion of the product can be subjectedto isomerization conditions, hydrogenation, hydrotreatment, and/orhydrofinishing conditions.

As used herein, the term “biodiesel” means a biofuel that can be asubstitute of diesel, which is derived from petroleum. Biodiesel can beused in internal combustion diesel engines in either a pure form, whichis referred to as neat biodiesel, or as a mixture in any concentrationwith petroleum-based diesel. In one embodiment, biodiesel can includeesters or hydrocarbons, such as aldehydes, alkanes, or alkenes.

As used herein, the term “biofuel” refers to any fuel derived frombiomass, biomass derivatives, or other biological sources. Biofuels canbe substituted for petroleum based fuels. For example, biofuels areinclusive of transportation fuels (e.g., gasoline, diesel, jet fuel,etc.), heating fuels, and electricity-generating fuels. Biofuels are arenewable energy source.

As used herein, the term “biomass” refers to a carbon source derivedfrom biological material. Biomass can be converted into a biofuel. Oneexemplary source of biomass is plant matter. For example, corn, sugarcane, or switchgrass can be used as biomass. Another non-limitingexample of biomass is animal matter, for example cow manure. Biomassalso includes waste products from industry, agriculture, forestry, andhouseholds. Examples of such waste products that can be used as biomassare fermentation waste, straw, lumber, sewage, garbage, and foodleftovers. Biomass also includes sources of carbon, such ascarbohydrates (e.g., monosaccharides, disaccharides, orpolysaccharides).

As used herein, the phrase “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).These include, for example, various monosaccharides, such as glucose,fructose, mannose, and galactose; oligosaccharides, such asfructo-oligosaccharide and galacto-oligosaccharide; polysaccharides suchas xylose and arabinose; disaccharides, such as sucrose, maltose, andturanose; cellulosic material, such as methyl cellulose and sodiumcarboxymethyl cellulose; saturated or unsaturated fatty acid esters,such as succinate, lactate, and acetate; alcohols, such as methanol,ethanol, propanol, or mixtures thereof. The carbon source can also be aproduct of photosynthesis, including, but not limited to, glucose. Apreferred carbon source is biomass. Another preferred carbon source isglucose.

As used herein, the term “conditions sufficient to allow expression”means any conditions that allow a host cell to produce a desiredproduct, such as a polypeptide, fatty acid, or its derives describedherein. Suitable conditions include, for example, fermentationconditions. Fermentation conditions can comprise many parameters, suchas temperature ranges, levels of aeration, and media composition. Eachof these conditions, individually and in combination, allows the hostcell to grow. Exemplary culture media include broths or gels. Generally,the medium includes a carbon source, such as glucose, fructose,cellulose, or the like, that can be metabolized by a host cell directly.In addition, enzymes can be used in the medium to facilitate themobilization (e.g., the depolymerization of starch or cellulose tofermentable sugars) and subsequent metabolism of the carbon source.

As used herein, “conditions that permit product production” refers toany fermentation conditions that allow a production host to produce adesired product, such as fatty acid or fatty acid derivatives (e.g.,fatty acids, hydrocarbons, fatty alcohols, waxes, or fatty esters).Fermentation conditions usually comprise many parameters. Exemplaryconditions include, but are not limited to, temperature ranges, levelsof aeration, and media composition. Each of these conditions,individually and/or in combination, allows the production host to grow.

To determine if conditions are sufficient to allow expression, a hostcell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48hours. During and/or after culturing, samples can be obtained andanalyzed to determine if the conditions allow expression. For example,the host cells in the sample or the medium in which the host cells weregrown can be tested for the presence of a desired product. When testingfor the presence of a product, assays, such as, but not limited to, TLC,HPLC, GC/FID, GC/MS, LC/MS, MS, can be used.

As used herein, the term “fatty acid” means a carboxylic acid having theformula RCOOH. R represents an aliphatic group, preferably an alkylgroup. R can comprise between about 4 and about 22 carbon atoms. Fattyacids can be saturated, monounsaturated, or polyunsaturated. In apreferred embodiment, the fatty acid is made from a fatty acidbiosynthetic pathway.

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acids. The fatty acidbiosynthetic pathway includes fatty acid enzymes that can be engineered,as described herein, to produce fatty acids, and in some embodiments canbe expressed with additional enzymes to produce fatty acids havingdesired carbon chain characteristics.

As used herein, the term “fatty acid derivative” means products made inpart from the fatty acid biosynthetic pathway of the production hostorganism. Fatty acid derivative also includes products made in part fromacyl-ACP or acyl-ACP derivatives. The fatty acid biosynthetic pathwayincludes fatty acid synthase enzymes which can be engineered asdescribed herein to produce fatty acid derivatives, and in some examplescan be expressed with additional enzymes to produce fatty acidderivatives having desired carbon chain characteristics. Exemplary fattyacid derivatives include for example, fatty acids, acyl-CoAs, fattyaldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols,ketones, and esters (e.g., waxes, fatty acid esters, or fatty esters).

As used herein, a “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Thevector can be capable of autonomous replication or integrate into a hostDNA. Examples of the vector include a plasmid, cosmid, or viral vector.The vector of this invention includes a nucleic acid in a form suitablefor expression of the nucleic acid in a host cell. Preferably the vectorincludes one or more regulatory sequences operatively linked to thenucleic acid sequence to be expressed. A “regulatory sequence” includesvarious control elements, promoters, enhancers, and other expressioncontrol elements (e.g., polyadenylation signals). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence, as well as tissue-specific regulatory and/or induciblesequences. The design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, and the like.

As used herein “operably linked” or “operatively linked” means that aselected nucleotide sequence (e.g., encoding a polypeptide describedherein) is in proximity with a promoter to allow the promoter toregulate expression of the selected nucleotide sequence. In addition,the promoter is located upstream of the selected nucleotide sequence interms of the direction of transcription and translation. By “operablylinked” is meant that a nucleotide sequence and a regulatory sequence(s)are connected in such a way as to permit gene expression when theappropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequence(s).

As used herein, “control element” means a transcriptional controlelement. Control elements include promoters and enhancers. The term“promoter” or “promoter sequence” refers to a DNA sequence thatfunctions as a switch that activates the expression of a gene. If thegene is activated, it is said to be transcribed or participating intranscription. Transcription involves the synthesis of mRNA from thegene. A promoter, therefore, serves as a transcriptional regulatoryelement and also provides a site for initiation of transcription of thegene into mRNA. Control elements interact specifically with cellularproteins involved in transcription (Maniatis et al., Science 236:1237,1987).

A nucleic acid refers to a DNA molecule (e.g., a cDNA or genomic DNA),an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA or RNAanalog can be synthesized from nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded, but preferably isdouble-stranded DNA. An “isolated nucleic acid” is a nucleic acid thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid. The term therefore covers, for example, (a) a DNA whichhas the sequence of part of a naturally occurring genomic DNA moleculebut is not flanked by both of the coding sequences that flank that partof the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein. Specificallyexcluded from this definition are nucleic acids present in mixtures ofdifferent (i) DNA molecules, (ii) transfected cells, or (iii) cellclones, e.g., as these occur in a DNA library such as a cDNA or genomicDNA library. The nucleic acid described above can be used to express aprotein of this invention. For this purpose, one can operatively linkthe nucleic acid to suitable regulatory sequences to generate anexpression vector.

The terms “peptide,” “polypeptide,” and “protein” are used hereininterchangeably to describe the arrangement of amino acid residues in apolymer. A peptide, polypeptide, or protein can be composed of thestandard 20 naturally occurring amino acid, in addition to rare aminoacids and synthetic amino acid analogs. They can be any chain of aminoacids, regardless of length or post-translational modification (forexample, glycosylation or phosphorylation). The peptide, polypeptide, orprotein “of this invention” include recombinantly or syntheticallyproduced fusion versions having the particular domains or portions thatare soluble. The term also encompasses polypeptides that have an addedamino-terminal methionine (useful for expression in prokaryotic cells).

A “recombinant” peptide, polypeptide, or protein refers to a peptide,polypeptide, or protein produced by recombinant DNA techniques; i.e.,produced from cells transformed by an exogenous DNA construct encodingthe desired peptide. The term “recombinant” when used with reference,e.g., to a cell, or nucleic acid, protein, or vector, indicates that thecell, nucleic acid, protein or vector, has been modified by theintroduction of a heterologous nucleic acid or protein or the alterationof a native nucleic acid or protein, or that the cell is derived from acell so modified.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain) amino acid residues (e.g., conservedamino acid substitutions) or nucleotides to a second amino acid ornucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities.

EXAMPLE Genome-Scale FBA Modeling

The genome-scale model of E. coli, iAF1260 was used in this example.This model consisted of three parts. From m metabolites and n reactions,an m×n stoichiometric matrix S was formed, whose ijth element S_(ij) isthe stoichiometric coefficient of metabolite i in reaction j. The vectorof flux values of v, whose jth element v_(j) is the flux though reactionj, were constrained by a lower bound vector a, whose jth element a_(j)is the lower bound of the flux through reaction j, an upper bound vectorb, whose jth element b_(j) was the upper bound of the flux throughreaction j. Finally, the linear objective is formed by multiplying thefluxes by an objective vector f, whose jth element f_(j) is the weightof reaction j in the biological objective. Thus, a biologically-optimalflux distribution is obtained by solving

max f′v

subject to Sv=0,

a≦v≦b.   (1)

Modeling of Metabolic Shunts

This example was limited to shunts of single reactants, i.e. eachreaction that the shunt catalyzes involves only a single reactant.However, the same principles could be applied to cases involving two ormore reactants in the shunt, though the modeling became substantiallymore complicated. For the thioesterase shunt, which catalyzes thereaction: fatty acyl-ACP_([c])→ACP_([c])+free fatty acid_([e]), thesingle reactant case sufficed.

Without loss of generality, it was supposed that the shunt catalyzes asingle reaction of the form

A→B.

In cases involving multiple reactions (as is the case for thethioesterase shunt, since there are multiple fatty acyl-ACP compounds iniAF1260), the transformation described below can be applied to each ofthe reactions in sequence. The number of products for the reaction isinconsequential.

Let the flux of this reaction be w, and suppose that there are Kreactions, v₁, v₂, . . . , v_(K) in the model that involve A as aproduct and L reactions v_(K+1), v_(K+2), . . . , v_(K+L) that involve Aas a reactant. The shunt was modeled by ensuring that some fraction α ofthe flux passing through A is forced through the shunt. Specifically,the following constraint was imposed

$\begin{matrix}{w \geq {\alpha {\left\{ {{\sum\limits_{k = 1}^{K}\; {\max \left( {v_{k},0} \right)}} + {\sum\limits_{k = {K + 1}}^{L}\; {\max \left( {{- v_{k}},0} \right)}}} \right\}.}}} & (2)\end{matrix}$

This constraint was enforced using the following linear constraints:

u _(k) =v _(k) +s _(k) , k=1, . . . , K,

u _(k) =−v _(k) +s _(k) , k=K+1, . . . , K+L,

w=Σ _(k=1) ^(K+L) αu _(k),

u_(k)≧0, ∀k,

s_(k)≧0, ∀k,   (3)

which can be incorporated into the constraints of (1) to obtain a linearoptimization problem that involves the shunt. In addition, the variablesu_(k) and s_(k) can be associated with reactions and interpreted asfluxes, allowing the specific form of problem (1) to be used with asuitably defined stoichiometric matrix S. As a consequence, the approachused by GDLS (Lun et al. 2009 Molecular Systems Biology 5: 296) wasapplied to identify genetic design strategies with favorable values forthe synthetic objective. The results were reported in Table 1 (α:=0.2)

TABLE 1 Genetic design strategies for fatty acid overproduction in E.coli K-12 MG1655 with thioesterase shunt. Number of Biomass flux Freefatty acid flux Genotype knockouts (h⁻¹) (mmol gDW⁻¹ h⁻¹) WT ′tesA 00.837675 0.336305 ΔfadE ′tesA 1 0.828200 0.397012 Δrpe ΔsgcE ΔtalAB 20.592367 0.552849 ′tesA

In sum, the analysis was started with iAF1260 (Feist et al. 2007Molecular Systems Biology 3: 121), the most recent and most detailed ofthe published FBA models of E. coli K-12 MG1655. Then, added to thismodel, was a component that modeled the thioesterase shunt (see above).Briefly, the added component forced a certain fraction of the fluxpassing through a fatty acyl-ACP compound in the model through thefollowing reaction:

fatty acyl-ACP_([c])→ACP_([c])+free fatty acid_([e]),

where the chain length of the free fatty acid is appropriate for thespecific fatty acyl-ACP involved in the reaction, and the subscriptsindicate the cellular compartment of the compound, with c representingthe cytoplasm, and e representing extracellular space.

Using GDLS, it was found that the optimal genetic design strategyinvolving one knockout was to knock out the fadE gene and that involvingtwo knockouts was to knock out the isozymes rpe/sgcE and talAB (seeTable 1). Of these strategies, one had already been previouslyexperimentally implemented. Specifically, the fadE knockout coupled withthe 'tesA thioesterase was shown to more than triple the yield of freefatty acids compared to wild-type E. coli DH1 with the 'tesAthioesterase, increasing the yield from 4% of the theoretical limit to14% Steen et al. 2010 Nature 463(7280): 559-562.

An E. coli strain bearing the two knockout strategy involving rpe/sgcEand talAB was produced and tested for its fatty acid production. GDLSalso produced other genetic strategies involving two or more knockouts.

In one example, strains of E. coli K-12 MG1655 bearing the fadE knockoutand the rpe/sgcE and talAB knockouts were generated. The fatty acidyield from these strains on a two-day fermentation on 2% (w/v) glucoseis summarized in Table 2. It was found that both the ΔfadE 'tesA and theΔrpe ΔsgcE ΔtalAB 'tesA strains show significantly increased fatty acidyield over wild type (7.1-fold and 3.9-fold respectively). Theabove-described modeling further predicts that the combination of allfive knockouts (i.e. fadE, rpe, sgcE, talA and talB) has greater fattyacid yield than the two knockout strains tested so far.

TABLE 2 Yield of fatty acids of mutant strains of E. coli MG1655 K-12.Yield (mg L⁻¹) Δrpe ΔsgcE WT WT ′tesA ΔfadE ′tesA ΔtalAB ′tesA Std. Std.Std. Std. Fatty acid Mean dev. Mean dev. Mean dev. Mean dev. C8:0 0.00.0 0.6 0.6 11.2 1.3 5.8 0.1 C10:0 0.0 0.0 0.0 0.0 2.3 0.4 1.1 0.0 C12:00.0 0.0 0.3 0.6 62.1 7.2 34.3 0.9 C14:0 3.5 0.9 3.1 0.8 119.7 13.1 73.32.1 C16:0 30.7 0.5 25.5 7.3 39.5 2.9 16.4 0.6 C16:1 0.0 0.0 0.3 0.5 34.32.7 20.2 1.3 C18:0 0.5 0.7 0.4 0.4 19.1 1.4 9.3 2.8 C18:1 0.4 0.5 0.30.5 2.2 0.2 0.4 0.3 C18:? 4.5 2.5 1.7 0.1 0.2 0.3 0.0 0.0 C18:2 0.0 0.00.0 0.0 0.3 0.6 0.0 0.0 C20:0 1.3 1.8 1.6 1.4 0.8 1.3 0.0 0.0 Total 40.96.9 33.8 12.3 291.8 31.4 160.7 8.2 Note: The mean and standard deviationare for three biological replicates of each strain, except WT, for whichthere were two replicates.

The foregoing example and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. All publications cited herein arehereby incorporated by reference in their entirety. As will be readilyappreciated, numerous variations and combinations of the features setforth above can be utilized without departing from the present inventionas set forth in the claims. Such variations are not regarded as adeparture from the scope of the invention, and all such variations areintended to be included within the scope of the following claims.

1. A method for constructing a model for identifying a target gene for agenetic alteration, the method comprising: obtaining a genome-scaleflux-balance analysis (FBA) model; adding to the model a component thatmodels a thioesterase shunt, wherein the added component forces afraction of the flux passing through a fatty acyl-ACP compound in themodel through the following reaction: fatty acyl-ACP[c]→ACP[c]+freefatty acid[e], wherein [c] represents the cytoplasm compartment and [e]represents extracellular space; obtaining a biological optimal fluxdistribution; and displaying a record comprising a gene returned by themodel, whereby the gene is the target for the genetic alteration,wherein an engineered microbe having the genetic alteration produces afatty acid at a level higher than a control microbe that is identical tothe engineered microbe except that the control microbe lacks the geneticalteration.
 2. A method for identifying a target for a geneticalteration, comprising obtaining one or more genes using the modelconstructed by the method of claim 1, wherein an engineered microbehaving the genetic alteration produces a fatty acid at a level higherthan a control microbe that is identical to the engineered microbeexcept that the control microbe lacks the genetic alteration.
 3. Themethod of claim 1, wherein the microbe is E. coli.
 4. The method ofclaim 1, wherein the model is an iAF1260 model.
 5. The method of claim1, wherein the biological optimal flux distribution is obtained by aGenetic Design through Local Search (GDLS).
 6. The method of claim 1,wherein the genetic alteration is an alteration of two or more genes. 7.The method of claim 1, wherein the genetic alteration is a knockout of agene.
 8. A machine-readable medium for carrying out the method of claim1, comprising machine-readable instructions encoded thereon which, whenexecuted by a processor, cause a machine having or linked to theprocessor to execute the method.
 9. A machine-readable medium on whichis stored a database capable of configuring a computer to respond toqueries based on a plurality of records belonging to the database, eachof the records comprising a genotype value that identifies a genotype ofa microbe having one or more genetic alterations; a gene value thatidentifies a gene having a genetic alteration; a fatty acid value thatidentifies a fatty acid; a biomass flux value that identifies a biomassflux; and a free fatty acid flux value that identifies a fatty acidflux, wherein the biomass flux and fatty acid flux are obtained usingthe method of claim
 1. 10. A computer system comprising themachine-readable medium of claim 8, and a user interface capable ofreceiving data and displaying the record.
 11. An isolated cell, thatlacks a functional gene, wherein the gene is selected from the groupconsisting of fadE, rpe, sgcE, talA, and talB, and a homologue thereof.12. The isolated cell of claim 11, wherein the cell lacks functionalfadE gene.
 13. The isolated cell, of claim 11, wherein the cell lacksone or more of the functional rpe, sgcE, talA, and talB genes.
 14. Theisolated cell of claim 11, wherein the cell is an E. coli cell.
 15. Theisolated cell of claim 14, wherein the cell is an E. coli K-12 MG1665cell.
 16. The isolated cell of claim 11, wherein the cell expresses athioesterase.
 17. A method of producing a fatty acid, comprising,culturing the cell of claim 11 in a culture under conditions allowingproducing of the fatty acid by the cell, and obtaining the fatty acidfrom the culture.
 18. A method of forming a hydrocarbon product,comprising obtaining fatty acids; performing thermal decarboxylation onsaid fatty acids to form a thermal decarboxylation product stream;hydrocracking the thermal decarboxylation product stream, and isolatinga product in the gasoline, jet, or diesel fuel range, wherein at least aportion of the fatty acids are derived from the cell of claim
 11. 19. Amethod of forming a hydrocarbon product, comprising obtaining fattyacids; performing thermal decarboxylation on said fatty acids to form athermal decarboxylation product stream; hydrocracking the thermaldecarboxylation product stream, and isolating a product in the gasoline,jet, or diesel fuel range, wherein at least a portion of the fatty acidsare derived from a cell that lacks a functional gene, wherein the geneis selected from the group consisting of fadE, rpe, sgcE, talA, andtalB, and a homologue thereof, wherein the fatty acids are obtainedaccording to the method of claim
 17. 20. The method of claim 18, whereinthe product is in the jet range.
 21. The method of claim 18, wherein allor a portion of the product is subjected to isomerization conditions.22. The method of claim 18, wherein all or a portion of the product issubjected to hydrogenation, hydrotreatment, and/or hydrofinishingconditions.
 23. The isolated cell of claim 16, wherein the thioesteraseis selected from the group consisting of tesA and 'tesA.